Multiplex chromogenic immunohistochemistry to stain and analyze paraffin tissue sections from the mouse or human

Summary Here we describe a multiplex chromogenic immunohistochemistry platform to stain and analyze two markers in paraffin tissue sections from mouse or human. The basis of the protocol is a series of stripping and re-probing steps with subsequent image analysis, which allows the user to perform multiplex imaging in a reliable and affordable manner. Here, we describe specific usage to assess the levels of PD-L1 in tumor-associated macrophages. We have used different antibodies and assessed this protocol for up to five consecutive antibodies per slide. For complete details on the use and execution of this protocol, please refer to Orgaz et al. (2020).1

Note: Hydrogen peroxide 30% stock bottle is stored at RT. Once prepared fill in two staining slide jars at RT under the fume hood.
Note: Mix to dissolve and adjust pH to 8.4 using concentrated HCl and then add 1 mL Tween 20.
Store this solution at RT. Dilute 1:10 with distilled water before use and adjust pH if necessary.
Alternatives: Can be used commercial wash buffer (103 Dako Wash Buffer, Agilent).

STEP-BY-STEP METHOD DETAILS
Rehydration and antigen retrieval round 1

Timing: 1-2 h
This step describes how to rehydrate tissue sections and perform antigen retrieval using a pressure cooker (See Figure 1).
1. Make sure slides are marked with appropriate information.
Note: Pencil is recommended for labelling the slides. Pens can be washed away due to subsequent steps using alcohols. Alternatively, pathology services use histology printer slides. 7. Use pressure cooker for heat-induced antigen retrieval (HIAR).
Alternatives: A water bath or microwave are also reliable methods for HIAR.
a. Switch on hotplate and set to full temperature (400 C) by turning the dial to maximum.
CRITICAL: For safety reasons, this protocol has been adapted for a conventional pressure cooker with a laboratory hot plate. Electrical pressure cookers can also be used.
b. Prepare the Antigen-retrieval buffer. See recipe in materials and equipment section ''Citrate antigen retrieval''. c. Replace the lid, but do not lock. d. Allow solution to warm until boiling (10 min). e. Carefully lower the rehydrated histological slides into the boiling solution. f. Replace lid and lock and set pressure selector to the highest-pressure symbol. g. Once the red pressure indicator has risen and a steady flow of steam is escaping from the outlet valve, start timing for 10 min. h. After 10 min, switch off the hotplate using the dial, carefully remove the pressure cooker and transfer to a sink.
Note: Changes in concentration, pH and Formalin exposure will introduce variability. Some epitopes are formalin-resistant while others undergo substantial changes (formalin-sensitive).
Alternatives: Citrate buffer (pH 6.0) is the most used solution for HIAR. This protocol uses citric buffer for antigen retrieval. Alternatively, EDTA (pH 8.0), Tris-EDTA (pH 9.0), and Tris (pH 10.0) buffers can be used. The pH of the buffers is an essential factor for antigen refolding or for antigenic determinant exposure. pH 8-9 is suitable for most antigens, although certain nuclear antigens show optimal staining at pH. 5 Unless the antigen retrieval method is stated on the antibody datasheet, the optimal method for each antigen must be tested experimentally.
CRITICAL: Enzymatic digestion such as proteinase K, trypsin and pepsin are not suitable for the current IHC multiplex protocol. Enzymatic digestion compromises the detection of epitopes that are suitable for HIAR. For instance, proteinase K is recommended when using anti-F4/80 antibodies, while poor antigen quality after consecutive anti-CD206 staining using pressure cooker antigen retrieval will lead to negative staining.
8. Lifting (or removing) the valve, pushing a button or turning a dial (depending on the pressure cooker). This method involves strong vapor evacuation.
CRITICAL: This release method does not cool down the pressure cooker like the cold-water release method. To avoid scalding injury, care must be taken when releasing the steam.
9. Once pressure cooker is open, allow the tissue slides to cool down in running tap water for 5 min. 10. Take out the slides from the pressure cooker and place them in a slide jar with ddH 2 0 ensuring the slides do not dry. The slides are now ready for subsequent staining.
Note: Tissue slides can be kept in water during 1-2 h. However, if longer periods are required, slides should be kept in TBS-Tween 20 (TBST).
CRITICAL: Reagents for dehydration and rehydration inside the staining jars can be kept under the fume hood at RT. However, it is important to change them once a week especially the H 2 O 2 endogenous peroxidase blocking solution.

OPEN ACCESS
Immunohistochemistry round 1

Timing: 3 h
This step describes how to detect antigen-antibody binding in tissue sections ( Figure 1).
Note: All the incubation processes should be performed under a humidified chamber in a staining tray at RT and covered to avoid slides drying out. To create a humidified chamber, add a piece of tissue paper covered in water at the bottom of the tray. Washes can be performed in histology staining jars.
11. Use ImmEdge Hydrophobic Barrier PAP Pen to draw a circle around a specimen on the slide. It will help to keep reagents localized on tissue specimens and prevents mixing of reagents when multiple sections are mounted on the same slide.
CRITICAL: PAP pen is considered as Cancer and Reproductive Harm. www.P65Warnings. ca.gov.

Transfer the slides into TBST.
Note: see recipe in materials and equipment section.
Alternatives: Commercial wash buffers with similar composition such as Dako wash buffer 103 can be used instead.
13. Remove unspecific staining by incubating the sections with 1%BSA-TBS for 30 min RT.
Alternatives: Normal serum can be used to block non-specific binding matching the species of the secondary antibody. Serum contains antibodies that bind to non-specific sites in the tissue or to Fc receptors. Pooled serum from healthy adult animals at 2.5% is commercially available. In this experiment, 2.5% Normal Goat Serum was used since the secondary antibody was raised in Goat.
Alternatives: Emerald diluent is commercially available -but 1% BSA-TBS can be used instead.
CRITICAL: This protocol was optimized for PD-L1 and CD206 detection in consecutive rounds. The antibody order needs to be optimized for each experiment.
Note: ImmPRESS reagents are sold as RTU solutions. Small biopsies or tissue sections such as mouse spleen are covered in 100 mL. However larger sections such as those from mouse primary tumors may require 300 mL to cover the entire section.
17. Rinse with TBST for 2 3 5 min at RT. CRITICAL: It is especially important to use PBS as diluent for VIP chromogen. ddH2O, antibody diluent or 1%BSA-TBS are not recommended. As specified by the manufacturer instructions, deionized water may contain inhibitors of the peroxidase reaction. Solutions containing sodium azide or other inhibitors of peroxidase activity should not be used when diluting the peroxidase substrate.
18. Apply Vector VIP HRP substrate chromogen for 10 min at RT.
Note: Previous published protocols use alcohol soluble chromogens for multiplex staining. VIP chromogen is stable in alcohol, but it is heat sensitive. Advantages of this chromogen: it is as sensitive as DAB and can be removed using regular antigen retrieval procedures such as pressure cooker, water bath or microwave. See troubleshooting section, problem 1.
19. Transfer slides to running tap water for 2 min at RT.
CRITICAL: Avoid direct contact from running tap water, it could displace the tissue attached to the glass slide.
CRITICAL: Always run appropriate positive and negative control sections with each staining batch. For the purposes of this protocol mouse spleen was used as positive control.
Note: Tissues where presence of a specific cellular population or antigen are known or expected can be used as positive controls in IHC. For unknown or undescribed antigens of interest https://www.proteinatlas.org/ can be an indicator. For immune cell populations in mice, mouse spleens are commonly used as positive controls. For human samples, tonsils are used due to easy access for pathology units. Appendix is also a widely used control since it is a tissue rich in inflammatory cells, epithelial cells and stroma. Negative controls are also recommended (no primary antibody or Isotype control antibody can be used). To evaluate specificity of new antibodies, cell lines transfected with siRNA targeting the gene/protein of interest can be used. Agar pellets of cells after RNAi transfections are embedded in paraffin and the same IHC protocol can be applied to confirm specific antibody binding. Efficiency of RNAi transfection needs to be confirmed by either qPCR or western blot.

Timing: 30 min
This step describes how to counterstain with Hematoxylin, dehydrate the tissues and mount with cover-slides ( Figure 1). This step describes how to image the stained sections using a high-capacity digital pathology slide scanner. In this case, we have used NanoZoomer s210 Digital slide scanner from Hamamatsu ( Figure 1).
30. Place the slides in the scanning tray and perform single or batch scanning, according to the number of slides in the experiment. 31. Set the Region of scan and place crosses in the focus points. Set 403 as resolution.
Pause point: If needed the slides can be stored and re-start Round 2 when desired. They can be used up to 6 times.
Note: Mounted stained tissue slides should be stored at RT in an airtight container. Slides can be reused even after two years, if the slides are kept dry.

Rehydration and antigen retrieval round 2
Timing: 1 h This step describes how to perform rehydration and antigen retrieval procedure in slides that have already been stained (Figure 1). 32. Place the slides from round 1 into Xylene jar for at least 12-24 h. 33. Remove the coverslip carefully. 34. Re-start the process of rehydration and antigen retrieval as in ''rehydration and antigen retrieval round 1'' section. This part of the protocol is the same as Round 1 (Figure 1).

Tissue scanning round 2
Timing: 30 min to 4 h This part of the protocol is the same as Round 1 (Figure 1).

Image alignment and pseudocolor image
Timing: 30 min per slide This step describes how to align images of two consecutive staining procedures and scans in the same tissue section. Furthermore, it creates a digital multiplex image that can be exported for quantification. To do so, QuPath 6 and FIJI ImageJ 7 were used (see Figure 2). 44. Create a project: Open QuPath and create a project with the images that need to be aligned. a. File> Project> Create a Project. Next, create a folder. Note: Store all the images in the same folder.
48. Image alignment of the exported ROIs. a. Open FIJI imageJ>File>New>TrakEM2 (blank). b. Create a folder where you will move the images you need to align ( Figure 3B).
Note: Whole slide images (WSI) are compressed in pyramidal stacks. Registration of WSI is complex. TrakEM2 does not allow registration of WSI.
49. Importing ROIs for alignment. a. Drag the folder (should start with trackem2.) with the two images, in this case PD-L1 and CD206 from the same region to the TrakEM2 plug-in. 50. Set up the alignment tools. Once the folder containing the images is dragged, the plug-in will ask type of Directory. a. Press stack. Next, the slide separation by default 1.   Figure 3D). 55. Create a pseudo-color composite image. Following the previous process, a new stack image will be generated. a. Go to ''Image''> ''Color''> ''Channel Tool.'' Channel to ''Color'' mode. For ''Channel 1'' go to ''More'', select color ''Red'', press ''More'' again and ''Edit LUT'' and ''Invert.'' b. Perform the same step in the Channel 2 and 3 using color Green and Blue accordingly. c. Finally Change ''Color'' mode to ''Composite''. Save the image in Tiff format ( Figure 3E and 3F).

Image analysis and quantification
Timing: 1-2 h for project In the example below, 5555 murine melanoma cells derived from the BRAF-V600E mouse melanoma model 8 were selected for their lack of response to anti-PD-1 in vivo (anti-PD-1/NR). 1 million 5555-anti-PD-1/NR cells were injected subcutaneously into 7-week-old C57BL/6J female mice. Tumors were grown for up to 27 days ( Figure 4A). Tumors were excised and fixed. The combination therapy of anti-PD1 plus ROCK inhibitor resulted in higher number of regressions ( Figure 4B), and IHC was performed to assess effects on macrophage markers ( Figure 4C).
Note: Due to strict limits for tumor volume in the UK animal license animals were culled when tumor volumes reached maximum size allowed or before any signs of skin ulceration were detected (redness, glossy skin). This step describes how to analyze images using QuPath of the ROIs alignment and process them using ImageJ (see Figure 4D-4F).
56. Create a project on Qupath. 57. Import the composite images from all the cases. 58. Change image type to ''Fluorescence'' Note: When importing the composite image ( Figure 4D) you need to assign the colors manually. ''Click'' in brightness and contrast icon and add a color for each channel manually. In our example, we used Channel 1: Blue, Channel 2: Green and Channel 3: Red.

Create annotations in all the cases.
CRITICAL: Before any segmentation or analysis, you need to generate annotation in the region of interest.
Note: Depending on antigen of interest subcellular localization, the score compartment should be optimized (Cell, Cytoplasm or Nuclear). Intensity thresholds should be adapted to the specific staining. Percentage of positivity instead of Histoscore can also be used as a measure by clicking ''single threshold''.
Note: For quantification purposes we use Histoscore instead of positive percentage since it provides semiquantitative information. Histoscore or H-score is used commonly in quantitative pathology. Hscore is calculated combining the intensity of staining (graded as: 0, nonstaining; 1, weak; 2, median; or 3, strong) and the percentage of positive cells. The range of scores is from 0 to 300.
CRITICAL: Default settings may allow cell detection, but this step may require some optimization. See troubleshooting section, problem 6.

Automation and batch analysis
Scripts can be used for Color deconvolution in FIJI and Image analysis in QuPath.
63. Creating macros in Fiji-ImageJ. Use recording tool in macros section. Go to Plugins>Macros> Record. Note: Commands are ordered by chronological order. If the last Positive Cell Detection is the definitive, remove all previous commands (see Figure 5B).
66. Create script. Store it and run it for the entire project. For this analysis the script is the following:

EXPECTED OUTCOMES
After performing stripping and re-reprobing and following the sections ''image alignment and pseudocolor image'' the user will be able to visualize the image as shown in Figure 3F. Merged images are uploaded in Qupath as described in ''image analysis and quantification''. After following the steps provides the user should be able to obtain quantitative information regarding the.

LIMITATIONS
Even if this is a robust, affordable and accessible pipeline, this protocol has several limitations: 1) Time consuming. Every marker has to be stained separately, which takes in average 6 h for staining plus scanning (time depends on the number of samples). Therefore, this protocol takes longer in comparison with simultaneous staining of several markers. A full protocol multiplexing 4-5 antibodies can take up to a week. However, there are no limitations in the host species of the primary antibody and cross-reactivity and no limitations with overlapping colors. 2) Tissue integrity. Tissue sections tolerate a high number of consecutive times in a pressure cooker (up to 8 rounds tested). However, detecting matrix proteins in desmoplastic or fibrotic tissues can be challenging. The order in which the primary antibodies are used needs to be optimized. For instance, it is advisable to use antibodies that recognize matrix-related proteins in the two first rounds. 3) Antigen retrieval. Other antigen retrieval methods have been tested such as microwave, water bath and enzyme digestion. The pressure cooker performs better than these methods because it can reach high-temperatures in short periods of time maintaining tissue integrity for further rounds. However, enzyme-based antigen retrieval such as pepsin, proteinase K, does not tolerate the ''stripping and re-probing'' method. Other antigens may also be affected using this method and giving negative results in the following rounds. 4) Montage and Image analysis. Image alignment using the proposed method is very laborious, especially if the user is working with high number of samples. Also, automation is limited and only possible for a few steps (see section automation and batch analysis). Presence of different intensity patterns/intensity in Immunohistochemical staining.

Potential solution
Changing from DAB to VIP chromogen may lead to variation in the intensity. When using an antibody for the first time, include positive and negative controls and parallel staining with DAB. Antibody concentrations need to be carefully titrated.

Problem 2
Chromogen remaining from previous round after HIAR.

Potential solution
Presence of chromogen (VIP) after HIAR in consecutive rounds might be due to suboptimal antigen retrieval or suboptimal optimization of previous round. Standard time for HIAR (10 min) needs to be prolonged for some antigens. Too intense staining due to saturation with primary antibody or exposure time with chromogen will affect the stripping process. Background-to-signal ratio needs optimization prior to cyclic IHC.

Problem 3
Lack of staining in consecutive rounds.

Potential solution
Tissues are sensitive to rounds of heat-induced antigen retrieval. Some antigens may no longer be detected. This is one the main benefits of using a pressure cooker since heat can be applied for very short periods of time. Soft-tissues or tissues rich in extracellular matrix (ECM) are more sensitive.
When using a panel of different antibodies, antibody order needs to be optimized. Very robust antigens such as pan-cytokeratin, ki67 among others should be added in the latest rounds.

Problem 4
Automatic image alignment not working.

Potential solution
TrackEM2 in FIJI-ImageJ also allows for supervised image alignment. Press ''right click'' in the mouse, go to ''align''>''Align layers manually with landmarks''. As shown in Figure 6, landmarks by similarity need to be created in the consecutive images. Important: same mark needs to be clearly labelled for both sections (see Figure 6).

Problem 5
Color deconvolution adjustment. Color deconvolution setting may need changes if the chromogens are not properly separated.

Potential solution
Here we show a step-by-step process using ImageJ to set up a custom color deconvolution using a control ROI from same experiment as template. Instead of selecting H AEC or H DAB, we select from ROI in the color deconvolution window. Follow ImageJ instructions and create a square where the chromogen is present, it will be Color 1. Create a second square where Chromogen 2 is present (this case nucleus, blue). For Color 3 just press 3 ''right click'' if your image has two colors like Hematoxylin and VIP or DAB.
To apply the same setting in the following images from same experiment, go to color deconvolution and select User Values. Fill with the values obtained from control image (highlighted in red) in Figure 7.

Problem 6
Cell segmentation issues.

Potential solution
The user may need to change some parameters according to the tissue or to cell composition. Important nuclear parameters are ''Background radius'', ''Median filter radius'', and ''sigma''. Background radius subtracts a background value from every pixel on the image and after applies a threshold. In case of many nuclei clustered together, increasing background radius number aids correct nuclei segmentation. Median filter radius and sigma can be useful parameters to remove fragmented nuclei from analysis (see Figure 8). In our case, default settings were amended with some custom values to allow optimal cell segmentation ( Figure 8).

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Prof Victoria Sanz-Moreno (v.sanz-moreno@qmul.ac.uk).

Materials availability
No materials were newly generated for this protocol. All materials mentioned above are commercially available.
Data and code availability Dataset used were published in JL Orgaz., et al. Cancer Cell 2020. 1 Codes used are posted in section above ''image analysis and quantification''.